H₂O₂-induced dilation in human coronary arterioles: Role of protein kinase G dimerization and large-conductance Ca²⁺-activated K⁺ channel activation

Abstract

Rationale

Hydrogen peroxide (H₂O₂) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H₂O₂ elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H₂O₂ involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-Iα, thereby modulating their activities.

Objective

Here we hypothesize that H₂O₂ dilates HCAs through direct oxidation and activation of PKG-Iα leading to the opening of the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel and subsequent smooth muscle hyperpolarization.

Methods and Results

Flow and H₂O₂ induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BK_Ca channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H₂O₂-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H₂O₂ elicited dose-dependent, reversible dimerization of PKG-Iα, and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K⁺ current with a unitary conductance of 246-pS in freshly isolated coronary SMCs. Addition of H₂O₂ into the bath solution significantly increased the probability of BK_Ca single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-Iα inhibitor DT-2. H₂O₂ exhibited an attenuated stimulatory effect on BK_Ca channel open probability in inside-out membrane patches.

Conclusions

H₂O₂ dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-Iα and subsequent opening of smooth muscle BK_Ca channels.

Introduction

Hydrogen peroxide (H₂O₂), a membrane permeable and relatively stable reactive oxygen species (ROS), has emerged as an important signaling molecule in the regulation of physiological and pathophysiological processes in vascular cells.^1–3 Our recent studies indicate that, in human coronary arterioles (HCAs), H₂O₂ serves as a key endothelium-derived hyperpolarizing factor (EDHF) responsible for both flow-mediated dilation (FMD) and to a lesser extent agonist-induced dilation.^4–6 As an EDHF, H₂O₂ elicits vasodilation through a mechanism involving smooth muscle Ca²⁺-activated K⁺ channel (K_Ca) activation and subsequent membrane hyperpolarization.⁶ Other studies have also demonstrated that H₂O₂ induces potent K_Ca-mediated smooth muscle hyperpolarization and relaxation in porcine coronary,^7–9 human¹⁰ and mouse mesenteric arteries.¹¹ Intriguingly, the role of H₂O₂ as a vasodilator factor in HCAs is more prominent in disease states [e.g., in coronary artery disease (CAD)], whereas other traditional factors [i.e., nitric oxide (NO) and prostacyclin (PGI₂)] play a more important role in vasodilation in the absence of CAD or its risk factors.⁶ Despite extensive data demonstrating the importance of H₂O₂ in mediating smooth muscle hyperpolarization and dilation, the precise mechanisms of action of H₂O₂ as an EDHF remain poorly understood. The lack of detailed analysis on the mechanism of action of H₂O₂ has raised some concerns regarding the proposed role of H₂O₂ as an EDHF.¹²

Accumulating evidence indicates that a conserved mechanism by which H₂O₂ activates intracellular signaling in cells involves the oxidation of key cysteine residues in its target proteins.¹³ A more recent study suggests that, via this mechanism, H₂O₂ induces dimerization and subsequent activation of protein kinase G (PKG)-Iα in vascular smooth muscle cells.¹⁴ PKG-Iα is an important regulator of activity of various smooth muscle K⁺ channels, including the large-conductance K_Ca (BK_Ca) channel.¹⁵ We hypothesized that H₂O₂ induces relaxation of HCAs through direct oxidation and activation of PKG-Iα leading to the opening of BK_Ca channels and subsequent smooth muscle hyperpolarization. Using an integrated approach comprising isolated vascular reactivity measurement, immunohistochemical and molecular biological analysis, and patch-clamp technique, we performed in-depth analysis of cellular mechanisms responsible for H₂O₂-induced dilation in HCAs. Our results demonstrate that BK_Ca channel activation plays a key role in flow- and H₂O₂-induced dilation of HCAs. We also demonstrate that the opening of BK_Ca channels requires an intermediate signaling event involving H₂O₂-induced protein dimerization of PKG-Iα.

Methods

A detailed Methods section is available in the Online Supplement at http://circres.ahajournals.org.

Tissue Acquisition

Fresh human right atrial appendages were obtained as discarded surgical specimens from patients undergoing cardiopulmonary bypass procedures as described previously.⁶ Patient demographic data are summarized in Online Table I.

Cell Culture

Human coronary artery smooth muscle cells (HCASMCs) were obtained from Lonza (Walkersville, MD), and cultured in full growth medium according to the manufacturer’s instructions. Cells between passages 4 and 6 were used for experiments.

Isometric Tension Recording

Coronary arterioles (≈100–200 µm) were carefully dissected from the endocardial surface of the atria, and mounted in multi-chamber wire myograph (model 610M, Danish Myo Technology) as previously described.^16,17 After contraction with endothelin-1, relaxation responses to cumulative concentrations of H₂O₂ (10⁻⁶–3×10⁻⁴ mol/L), spermine NONOate (10⁻⁷–3×10⁻⁵ mol/L), a NO donor, or 8-pCPT-cGMP (10⁻⁶–10⁻⁴ mol/L), a membrane-permeable analogue of guanosine 3’,5’-cyclic monophosphate (cGMP), were determined in the absence or presence of following inhibitors: iberiotoxin (IbTX; 100 nmol/L), a specific inhibitor of BK_Ca channels; catalase (1000 U/ml), a H₂O₂ metabolizing enzyme; ODQ (10 µmol/L), a selective inhibitor of soluble guanylate cyclase (sGC); Rp-8-Br-PET-cGMP (100 µmol/L), a competitive PKG inhibitor; or DT-2 (10 µmol/L), a specific, peptide inhibitor of PKG-Iα. To examine the role of smooth muscle hyperpolarization in H₂O₂-induced dilation, arteries were pre-constricted with high-K⁺ (80 mmol/L K⁺) Krebs (K-PSS). Vasodilator responses are expressed as percent maximal relaxation relative to endothelin-1 or K-PSS constriction, with 100% representing full relaxation to basal tension.

Videomicroscopy

Coronary arterioles (≈100–200 µm) were cannulated with 2 glass micropipettes, and the internal diameter of arterioles was measured with a video system.^6,16 Vessels were constricted with endothelin-1 to 30–50% of the baseline internal diameter. In studies of flow-induced dilation, flow was produced by changing the heights of two syringe reservoirs in equal and opposite directions to generate a pressure gradient.^6,16 Flow-mediated responses (5–100 cm H₂O) were examined before and after 30 min incubation with ODQ (10 µmol/L), Rp-8-Br-PET-cGMP (100 µmol/L), or iberiotoxin (IbTX, 100 nmol/L). At the end of each experiment, papaverine (10⁻⁴ mol/L), an endothelium-independent vasodilator, was added to determine the maximal internal diameter for normalization of dilator responses.

Enzymatic Isolation of Vascular Cells

Vascular endothelial cells (ECs) and SMCs were enzymatically dissociated from arteries as previously described.^16,18 Cells were placed on ice or at 4°C and used the same day.

Patch-Clamp Recording of K⁺ Currents

Single-channel K⁺ currents were recorded from cell-attached and excised inside-out membrane patches of freshly isolated coronary SMCs using the patch-clamp method as previously described.¹⁸ For both cell-attached and inside-out patches, the pipette solution contained (in mmol/L) 125 KCl, 1.8 CaCl₂, 1.0 MgCl₂, 10 ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 5 HEPES (pH 7.2), and the bath solution contained (in mmol/L) 125 KCl, 1.8 CaCl₂, 1.0 MgCl₂, 10 glucose, 10 EGTA, and 5 HEPES (pH 7.2). Channel currents were recorded for at least 3–6 min under control conditions and after treatment with H₂O₂ (10–100 µmol/L) in the absence or presence of paxilline (100 nmol/L, a specific and cell-permeable inhibitor of BK_Ca channels). Unless otherwise stated, all chemicals were applied to the bath through perfusion. To determine the role of PKG-Iα in H₂O₂-induced BK_Ca channel activation, cells were preincubated for 45–60 min with DT-2 (10 µmol/L) and the effect of H₂O₂ determined. Experiments were performed at room temperature.

RNA Extraction and RT-PCR

Total RNA from vascular tissues was extracted with TRIzol, and cDNA was synthesized. For freshly isolated vascular cells, RNA extraction was performed as previously reported.¹⁶ The cDNA was subjected to PCR amplification using a 38-cycle touch-down protocol with gene-specific primers.

Immunoblot Analysis

Protein samples (10–20 µg) were subjected to 10% SDS-PAGE. Membranes were blotted with a monoclonal mouse antibody specific to a conserved epitope of Slo1 (1:1000 dilution; NeuroMab L6/60 clone) or with a polyclonal rabbit anti-human PKG-I (1: 400 dilution; Abcam), followed by horseradish peroxidase-conjugated secondary antibodies.

Immunohistochemistry

Freshly dissected coronary arterioles were fixed with 10% formalin, embedded in paraffin wax, and cut into 4-µm sections. Sections were blocked for endogenous peroxidase and non-specific protein binding, and probed with a polyclonal rabbit antibody specific to human PKG-I (1:100 dilution; Abcam). For immunodetection, sections were incubated with a HRP-conjugated antibody, and then with a peroxidase-substrate solution.

Immunocytochemistry

Freshly isolated SMCs were fixed with 4% paraformaldehyde for 10 min, and then permeabilized with 0.1% saponin for 10 min. Cells were then incubated either with a polyclonal rabbit antibody specific to human PKG-I (1:50 dilution; Abcam), or a monoclonal mouse antibody specific to a conserved epitope of Slo1 (1:200 dilution; NeuroMab L6/60 clone), followed by an appropriate goat secondary antibody conjugated with Alexa Fluor 488 (Invitrogen). Images were immediately taken using an epi-fluorescence microscope (model Eclipse TE-200, Nikon) with a 60x (NA 1.40) oil objective.

Statistical Analysis

Data are presented as means ± SE. Significant differences between mean values were evaluated by Student t test or ANOVA followed by the Student-Newman-Keuls multiple-comparison test. To compare concentration-response between treatment groups, a two-way repeated measures ANOVA was used. P values of p<0.05 were considered statistically significant.

Results

Role of BK_Ca channels in H₂O₂-induced dilation of human coronary arterioles

To examine the mechanism of H₂O₂-induced dilation in HCAs, graded doses of H₂O₂ (10⁻⁶–3×10⁻⁴ mol/L) were applied to endothelium-intact and -denuded HCAs. H₂O₂ induced similar concentration-dependent dilation of both endothelium-intact (Figure 1A; dilation at 10⁻⁴ mol/L, 84±4%, n=13) and -denuded (Figure 1B; 85±8%, n=4) HCAs, indicating an endothelium-independent smooth muscle mechanism of dilation. The dilation was markedly inhibited by the specific BK_Ca channel blocker iberiotoxin (100 nmol/L) in both endothelium-intact (Figure 1A; 3±2%, n=9, P<0.05 versus control) and -denuded (Figure 1B; 19±10%, n=4, P<0.05 versus control) HCAs, implicating BK_Ca channels as the smooth muscle end-effectors of H₂O₂-induced relaxation. Consistent with previous data from our laboratory,⁶ dilation to H₂O₂ was completely abolished by high K⁺ (1±5%, n=7, P<0.05 versus control) as well as by 1,000 U/ml exogenous catalase (10±4%, n=3, P<0.05 versus control), a H₂O₂-metabolizing enzyme, confirming that this relaxation response is elicited through a K⁺ channel end-effector and is a H₂O₂-sensitive effect (Figure 1C).

Figure 1. Role of BK_Ca channels in H₂O₂-mediated dilation of human coronary arterioles.

H₂O₂ induced similar dose-dependent dilation of both endothelium-intact (A) and -denuded (B) arterioles. These relaxations were inhibited by 100 nmol/L iberiotoxin (IbTX), a BK_Ca channel blocker, and eliminated by high K⁺ (80 mmol/L) and exogenous catalase (1,000 U/ml) (C). n=3–13 vessels/each group; *P<0.05 vs. control.

Role of sGC and PKG-Iα in H₂O₂- and NO-mediated dilation of human coronary arterioles

To further examine the mechanism by which H₂O₂ induces smooth muscle hyperpolarization through BK_Ca channel activation, the role of the sGC-cGMP-PKG pathway in H₂O₂- and NO-mediated dilation was investigated. As illustrated in Figure 2A, H₂O₂ dose-dependently dilated HCAs (dilation at 10⁻⁴ mol/L, 82±3%, n=15). The dilation was attenuated by the PKG inhibitor Rp-8-Br-PET-cGMP (100 µmol/L) (5±6%, n=6, P<0.05 versus control) but not by ODQ (10 µmol/L), an inhibitor of sGC (61±8, n=9), indicating that H₂O₂ acts downstream of sGC at the level of PKG. DT-2, a peptide inhibitor of PKG-Iα, further confirmed the downstream action of this transferrable dilator agent, as the H₂O₂ -induced relaxation (81±6%, n=3) was significantly reduced by administration of 10 µmol/L DT-2 (13±9%, n=3, P<0.05 versus control) (data not shown). In contrast, the NO donor spermine NONOate-induced relaxation (dilation at 10⁻⁵ mol/L, 76±6%, n=11) were inhibited by both Rp-8-Br-PET-cGMP (16±1%, n=4, P<0.05 versus control) and ODQ (7±6%, n=4, P<0.05 versus control), signifying NO action upstream of both sGC and PKG (Figure 2B). Further, direct PKG activation by the stable cell-permeable cGMP analogue 8-pCPT-cGMP (10⁻⁶–10⁻⁴ mol/L) dilated HCAs in a concentration-dependent manner (dilation at 10⁻⁴ mol/L, 42±9%, n=7) as shown in Figure 2C. This response was blocked by iberiotoxin (15±6%, n=5, P<0.05 versus control), confirming a role for BK_Ca channels in PKG-induced smooth muscle hyperpolarization and relaxation. As summarized in Online Table II, no significant effect on baseline vascular tone was observed for ODQ, Rp-8-Br-PET-cGMP, and iberiotoxin, and similar dilation to the smooth muscle relaxant papaverine (100 µmol/L) was found in arterioles with or without pretreatment with these inhibitors.

Figure 2. Role of guanylate cyclase and PKG in H₂O₂- and NO-mediated relaxation of human coronary arterioles.

A: H₂O₂-induced relaxation responses were inhibited by the PKG inhibitor Rp-8-Br-PET-cGMP (100 µmol/L) but not by the soluble guanylate cyclase inhibitor ODQ (10 µmol/L). B: The NO donor spermine NONOate-induced relaxations that were inhibited by both Rp-8-Br-PET-cGMP and ODQ. C: The stable cGMP analogue 8-pCPT-cGMP dose-dependently dilated coronary arterioles, a response inhibited by the BK_Ca channel blocker iberiotoxin (100 nmol/L). n=4–15 vessels/each group; *P<0.05 vs. control.

H₂O₂-induced protein dimerization of PKG-Iα in human coronary artery smooth muscle cells

Recent evidence suggests that H₂O₂ directly activates the PKG isoform PKG-Iα but not PKG-Iβ by oxidizing a specific cysteine residue of PKG-Iα, leading to disulfide dimerization of this isoform.¹⁴ To elucidate the role of H₂O₂ in the dimerization and activation of PKG-Iα in HCAs, immunohistochemistry and Western blotting were used to assess protein expression of this kinase. Immunohistochemical analysis demonstrated expression of PKG-I protein in coronary arterioles, in particular within the smooth muscle cell layer (Figure 3A, left). This was further verified by Western blot, which revealed that under basal conditions, most of PKG-I exists as a 75 kDa monomer form (Figure 3A, right). Treatment of cultured coronary SMCs with H₂O₂ induced concentration-dependent dimerization of PKG-I, an effect which was abolished in the presence of the reducing agent β-mercaptoethanol. Given that only PKG-Iα undergoes disulfide dimerization in the presence of H₂O₂, these results indicate that PKG-I proteins detected in coronary SMCs are mostly the 1α isoform. Immunoblotting studies of cultured human vascular SMCs from multiple vascular beds using PKG-Iα- and PKG-Iβ-specific antibodies also revealed that PKG-Iα is the predominant isoform of PKG-I.¹⁹

Figure 3. H₂O₂-induced protein dimerization of PKG-I in human coronary artery smooth muscle cells.

A: The protein expression of PKG-I in HCAs was detected by immunohistochemical analysis (left panel; scale bar = 50 µm) and Western blot (right panel). Under basal conditions, PKG-I was primarily in monomeric form. B: Treatment of HCASMCs with H₂O₂ induced concentration-dependent dimerization of PKG-I which was blocked by the reducing agent β-mercaptoethanol (β-ME). Data are representative of 3 independent experiments. C: Immunofluorescence detected diffuse cytosolic expression of PKG-I under control conditions (upper). H₂O₂ (100 µmol/L) induced punctate expression of PKG-I along the plasma membrane (middle). Scale bar = 20 µm. Data are representative of 3 independent experiments with 5–10 cells/group/experiment.

Immunofluorescence was used to visualize the translocation of PKG-Iα in freshly isolated coronary artery smooth muscle cells. Diffuse cytosolic staining of PKG-Iα was observed under control conditions (Figure 3C, upper panel). Administration of 100 µmol/L H₂O₂ changed the staining pattern to one punctuated by expression along the plasma membrane (Figure 3C, middle panel), suggesting translocation of the protein from the cytosol to the plasma membrane upon activation by H₂O₂. Determination of plasma membrane/cytoplasmic ratio of PKG-Iα fluorescence confirmed the plasma membrane localization of this protein in H₂O₂-treated cells (1.30±0.04 versus 0.88±0.02 of control; P<0.05). The lack of signal in the preparations without primary antibodies (Figure 3C, lower) shows that this is an immunospecific response.

Expression of BK_Ca channels in coronary smooth muscle cells

BK_Ca channel expression is known to be modulated by disease,^20,21 but its expression in HCAs from patients with CAD is not clear. Therefore, the expression of BK_Ca channels in coronary SMCs of patients with and without CAD was examined. Expression of BK_Ca α-subunit was detected at both transcript (mRNA) (Figure 4A) and protein (Figure 4B) levels from patients with and without CAD. The average protein expression level of BK_Ca α-subunits showed no significant difference between patients with CAD and those without, although in 1–2 samples (out of 8) from CAD patients the level of BK_Ca channel expression was much higher than average. The expression of BK_Ca channels on the plasma membrane was further verified via immunofluorescence, which demonstrated BK_Ca channel immunoreactivity (in green) in smooth muscle cells of patients with CAD (Figure 4C, middle) or without CAD (Figure 4C, upper). Cell nuclei were stained in blue. Analysis of plasma membrane/cytoplasmic ratio of BK_Ca fluorescence showed a similar plasma membrane distribution of this protein in smooth muscle cells from patients with and without CAD (1.25±0.03 and 1.23±0.03, respectively; P>0.05). To examine the functional status of BK_Ca channels, single-channel activity and conductance were measured at different patch potentials using inside-out patches. As illustrated in Figure 4D (left), the channel open state probability (NPo) was enhanced with each sequential increase in patch potential. Channel openings were abolished by the addition to the bath of 100 nmol/L paxilline, a selective and cell-permeable BK_Ca channel blocker. The current-voltage relationship determined over the voltage range of −60 and +60 mV in 20-mV increments revealed a unitary conductance of 246 pS, and a reversal potential of 0 mV when recorded in symmetrical K⁺ (145 mmol/L) solutions (Figure 4D, right). These findings indicate that the 246-pS single-channel K⁺ currents recorded from HCA smooth muscle cell membranes display electrophysiological and pharmacological properties consistent with those of BK_Ca single-channel currents recorded from conduit coronary SMCs.²²

Figure 4. Expression of BK_Ca channels in coronary smooth muscle cells.

A: Expression of BK_Ca channel α-subunits was detected at mRNA level in freshly isolated SMCs from patients with and without CAD, as indicated by a representative image of RT-PCR analysis. B: BK_Ca α-subunit protein was expressed in coronary arterioles from patients with (patient no. in black) and without CAD (patient no. in gray). Lower, summarized data; n=8 and 9 for CAD and non CAD, respectively. C: Presence of BK_Ca channel α-subunit protein (green) is confirmed with immunocytochemistry using freshly dispersed SMCs. Cell nuclei are stained in blue. Scale bar = 20 µm. Data are representative of 3 independent experiments with 5–10 cells/ group/ experiment. D: In inside-out patches of freshly isolated SMCs from human coronary arterioles, an increase in membrane potential enhanced BK_Ca channel open probability (left) that was abolished by 100 nmol/L paxilline, a specific BK_Ca channel inhibitor. The current-voltage relationship revealed a unitary conductance of 246 pS with a reversal potential of 0 mV in symmetrical (145 mmol/L) K⁺ solutions (right). c, closed state. n=4 patches.

Effect of H₂O₂ on BK_Ca channel currents in isolated human coronary artery smooth muscle cells

To investigate the activation of BK_Ca channel currents by exogenously applied H₂O₂, BK_Ca single-channel currents were recorded in cell-attached or inside-out membrane patches of freshly isolated smooth muscle cells of HCAs using the patch clamp technique. In cell-attached patches, addition of 50 µmol/L H₂O₂ to the bath induced activation of BK_Ca single-channel currents (NPo, 0.0050±0.0017 before versus 0.0495±0.0147 after H₂O₂; n=11, respectively; P<0.05) that was subsequently blocked by the addition of 100 nmol/L paxilline to the bath (NPo, 0.0039± 0.0011, n=10, P<0.05 versus H₂O₂) (Figure 5A), indicating that H₂O₂ activates smooth muscle BK_Ca channels. The activation of BK_Ca channels by H₂O₂ was concentration-dependent in the concentration range of 10 to 100 µmol/L (data not shown). Compared to cell-attached patches, the effect of H₂O₂ on BK_Ca single-channel opening in inside-out membrane patches was markedly reduced (NPo, 0.0048±0.0007 before and 0.0085±0.0028 after; n=12, respectively; P > 0.05). Together, these results suggest that H₂O₂ activation of BK_Ca channels requires an intracellular second messenger signaling pathway. To determine whether PKG participates as an intracellular signaling molecule in the H₂O₂-induced BK_Ca activation, the effects of H₂O₂ on BK_Ca single-channel currents were measured in cell-attached patches in the presence of 10 µmol/L DT-2, a PKG-Iα inhibitor. As demonstrated in Figure 5C, inhibition of PKG-Iα by DT-2 attenuated the increase in BK_Ca single-channel activity elicited by H₂O₂ (NPo, 0.0077± 0.0026 before versus 0.0103 ± 0.0034 after; n=10, respectively; P > 0.05) to a level similar to those observed in the inside-out membrane patches lacking intracellular signaling systems.

Figure 5. Effect of H₂O₂ on BK_Ca channel currents in freshly isolated coronary arteriolar smooth muscle cells.

Using cell-attached patches (A), H₂O₂ (50 µmol/L) increased BK_Ca single-channel currents in a paxilline (100 nmol/L)-sensitive fashion. However, H₂O₂-induced BK_Ca activation was greatly diminished in single-channel recordings from inside-out patches which lack intracellular constituents (B). H₂O₂-activated BK_Ca single-channel currents recorded from cell-attached patches were reduced in the presence of 10 µmol/L DT-2, a PKG-Iα inhibitor (C). c, closed state; PP, patch potential. n=6–12 patches/each group; *P<0.05 vs. control, ^#P<0.05 vs. H₂O₂.

Role of sGC, PKG, and BK_Ca channels in human coronary arteriolar relaxation to flow

To verify the functional significance of the proposed signaling pathway, the roles of guanylate cyclase, PKG, and BK_Ca in FMD was examined. H₂O₂ has been demonstrated to be a key mediator of FMD in HCAs.^4,6 Pretreatment with the sGC inhibitor ODQ had no effect on FMD (dilation at 100-cm H₂O gradient, 62±8% versus 68±3% of control; n=3, respectively; P > 0.05) indicating no role for sGC (Figure 6A). In contrast, inhibition of PKG by Rp-8-Br-PET-cGMP significantly attenuated flow-mediated dilation (30±5% versus 58±3% of control; n=5, respectively; P<0.05), suggesting the involvement of downstream PKG in the relaxation response to increased flow. Furthermore, flow-induced dilation was significantly impaired in the presence of iberiotoxin (30±5% versus 67±4% of control; n=6, respectively; P<0.05), implicating a role for BK_Ca channel activity as an initiating mechanism of smooth muscle hyperpolarization and dilation. Treatment of arterioles with ODQ, Rp-8-Br-PET-cGMP and iberiotoxin did not significantly affect baseline vessel diameters (Online Table II).

Figure 6. Role of guanylate cyclase, PKG, and BK_Ca channels in human coronary arteriolar relaxation to flow.

Fluid flow induced dilation of human coronary arterioles was attenuated by the PKG inhibitor Rp-8-Br-PET-cGMP (B) and by the BK_Ca channel blocker iberiotoxin (C). The guanylate cyclase inhibitor ODQ, had no effect (A). n=3-6 vessels/each group; *P<0.05 vs. control.

Discussion

This study uncovers a novel mechanism of H₂O₂-mediated smooth muscle hyperpolarization and dilation in the human coronary microcirculation. The major new findings are threefold. First, exogenous H₂O₂-induced dilation of HCAs results from the opening of smooth muscle BK_Ca channels. Second, the opening of BK_Ca channels requires intracellular signaling through H₂O₂-induced dimerization and translocation of PKG-Iα. Third, shear stress, which releases endogenous endothelium-derived H₂O₂, dilates HCAs by activating a similar signaling pathway as exogenous H₂O₂. In sum, these data indicate that H₂O₂-induced protein dimerization and activation of PKG-Iα and subsequent opening of BK_Ca channels serves as an important mechanism responsible for H₂O₂-mediated dilation of HCAs. The findings of this study provide further support for the proposed role of H₂O₂ as a diffusible EDHF in the human coronary microvessels⁴ and may apply to other coronary stimuli that induce a H₂O₂-mediated dilation, such as metabolic coronary dilation.²³

PKG in H₂O₂-induced dilation

The role of cGMP as a second messenger for NO-induced vasodilation has been well established.²⁴ This mediator is synthesized in SMCs by sGC, a cytosolic enzyme activated by NO donors and endogenous NO released from ECs in response to vasodilators such as acetylcholine and bradykinin. Once generated, cGMP activates its downstream target PKG (type Iα and Iβ) in SMCs, followed by the initiation of a variety of cellular processes leading to smooth muscle relaxation.²⁵ Consistent with this general mechanism, both sGC and PKG inhibitors markedly reduced NO-mediated dilation in HCAs. In contrast, inhibition of PKG (specifically PKG-Iα) but not sGC markedly reduced H₂O₂-mediated dilation in HCAs, indicating that H₂O₂ acts downstream of sGC signaling and at the level of PKG-Iα. Such lack of inhibition by the sGC inhibitor ODQ has also been reported previously by our laboratory.⁶

The PKG-Iα and -Iβ are two splice-variant isoforms of PKG, differing in their N-terminus by ~100 amino acids. However, only the Iα isoform is redox sensitive and activated by H₂O₂ through disulfide dimerization.¹⁴ The activation of different targets within the cGMP-PKG pathway by H₂O₂ and NO is further supported by the findings that H₂O₂-mediated dilation was abolished by high K⁺, whereas NO-mediated dilation was only partially inhibited by high K⁺ (data not shown). It is generally agreed that PKG-I mediates smooth muscle relaxation through both Ca²⁺-dependent (e.g., K⁺ channel activation) and Ca²⁺-independent (e.g., changes in the activity of myosin light chain phosphatase) mechanisms, but two PKG-I isoforms may differ in their specific mechanisms of relaxation.²⁵ Therefore, it is possible that the selective activation of PKG-Iα but not PKG-Iβ contributes, at least partially, to H₂O₂-induced high K⁺-sensitive dilation. It is also interesting to note that H₂O₂ activates PKG-Iα through a marked (>10-fold) increase in the affinity (indicated by a decrease in K_m) for substrate, whereas cGMP activates PKG-Iα primarily by increasing its maximum velocity (V_max) without changes in the K_m for substrate.¹⁴ Thus, an increase in PKG-Iα affinity for selected substrates (e.g., K⁺ channels) may serve as another potential mechanism underlying high K⁺-sensitive dilation to H₂O₂. The (patho)physiological significance of the specific signaling initiated by H₂O₂ versus NO in vascular smooth muscle remains to be clarified.

It has been well demonstrated that H₂O₂ relaxes bovine pulmonary and coronary arteries primarily through sGC activation.^26–28 The mechanism by which H₂O₂ stimulates sGC activity has been associated with the formation of compound I, a short-lived oxidized intermediate form of catalase that occurs during the metabolism of H₂O₂ by this enzyme.²⁶ Pretreatment of the reaction mixture with the catalase inhibitor 3-amino-1,2,4-triazole (3-AT) reduces the stimulatory effect of catalase in the presence of H₂O₂ on sGC activity, further suggesting the role of catalase in H₂O₂-induced activation of sGC.²⁶ Despite the stimulatory effect on sGC by H₂O₂, H₂O₂-induced relaxation is resistant to the sGC inhibitor ODQ in both pulmonary and coronary arteries.²⁷ Therefore, the possible contribution of sGC in H₂O₂-induced dilation of human coronary arterioles can not be completely excluded. However, the effectiveness of ODQ as an inhibitor of sGC activation by H₂O₂ may depend on vascular beds studied; for example, ODQ inhibits a portion of H₂O₂-induced dilation in porcine coronary arteries.⁹ In addition, 3-AT does not affect H₂O₂-induced dilation in HCAs (authors’ unpublished observations), indicating that catalase or its derived compound I is not involved in the vasodilatory responses of H₂O₂.

K⁺ channels in H₂O₂-induced dilation

There is substantial evidence that H₂O₂ applied exogenously produces membrane hyperpolarization and relaxation of smooth muscle in several vascular beds.^{4–11,29–32} However, disparate results have been reported for the specific K⁺ channel(s) involved in H₂O₂-induced hyperpolarization or dilation, and the signaling cascade leading to K⁺ channel activation remains largely unexplored. Activation of ATP-sensitive K⁺ (K_ATP) channels has been implicated in H₂O₂-mediated dilation of porcine and cat cerebral arteries.^29,30 A number of studies indicate that BK_Ca channels play an important role in H₂O₂-induced dilation of porcine coronary arteries and arterioles,^7–9 whereas other studies show that voltage-gated K⁺ (Kv) channels but not BK_Ca channels contribute to the dilatory effect of H₂O₂ in canine and rat coronary arterioles.^31,32 By measuring both isolated vascular reactivity and single K⁺-channel activity, we demonstrate that H₂O₂-induced dilation of human coronary arterioles depends on increased opening of BK_Ca channels. In particular, our patch-clamp studies showed that H₂O₂ activated BK_Ca currents recorded from the cell-attached patches (intracellular components present) but not from inside-out patches (intracellular components absent) and that the stimulatory effect of H₂O₂ on BK_Ca activity was markedly reduced by the PKG-Iα inhibitor DT-2. Taken together, these data provided strong evidence that H₂O₂-induced BK_Ca channel opening requires intracellular signaling, i.e. the activation of PKG-Iα in smooth muscle cells.

Several recent studies indicate that BK_Ca channels are under complex but coordinated regulation by a variety of protein kinases, including protein kinase C (PKC), protein kinase A (PKA), and PKG, as well as by protein phosphatases including protein phosphatase 1.^33–35 Specifically, phosphorylation of S¹¹⁵¹ by PKC during receptor agonist stimulation renders the channel responsive to PKG but prevents its activation by PKA, whereas PKC phosphorylation of both S¹¹⁵¹ and S⁶⁹⁵ renders the channel insensitive to PKG and PKA. PKG sensitivity can be rescued by protein phosphatase 1, a BK_Ca-associated protein that is constitutively active against phosphorylation of S⁶⁹⁵. The balance between protein kinases and protein phosphatases may vary in different smooth muscle preparations, contributing to the sensitivity of BK_Ca channels and potentially other K⁺ channels to PKG and activators such as H₂O₂.

There are rather extensive data from experimental animal models indicating that different diseases can have divergent effects on the expression and/or channel activity of the vascular smooth muscle BK_Ca channel.²⁰ For example, an upregulation of BK_Ca channel expression associated with an increased K⁺ current and vasodilation has been reported in SHR and DOCA-salt-induced hypertensive rats.²⁰ In contrast, a loss of BK_Ca-mediated dilation with an unexpected increase in the expression of BK_Ca channel subunits has been described in the coronary microcirculation using a swine model of metabolic syndrome.²¹ In the present study, we found that there is no significant difference in the protein expression level of coronary smooth muscle BK_Ca α-subunits between patients with CAD and those without, and more importantly, the BK_Ca channel is functional and mediates the dilation of coronary arterioles to H₂O₂ and flow in disease. Thus, our data further underscore the complexity of BK_Ca channel regulation by cardiovascular pathologies.²⁰

It remains to be determined whether flow- and H₂O₂-induced activation of BK_Ca channels in HCAs represents a compensatory pathway for the loss or impaired function of other K⁺ channels in the presence of CAD and other risk factors. As discussed above, H₂O₂-induced dilation is mediated by Kv channels in the coronary microcirculation of dogs and rats.^31,32 Although the involvement of different K⁺ channels in H₂O₂-induced dilation in the human versus animals may simply reflect species-specific differences, there is evidence that Kv channels may be importantly involved in H₂O₂-induced dilation in relatively healthy subjects without preexisting CAD or many other cardiovascular disorders (author’s unpublished observations). Because functional BK_Ca channels are present in subjects both with and without CAD and risk factors, the activation of BK_Ca channel-mediated dilation in CAD may result from the modulation of signaling pathways (e.g. protein phosphorylation) rather than from the change of the number of functional BK_Ca channels.

Interestingly, when applied to the cytosolic side of an excised membrane patch in human embryonic kidney (HEK)-293 cells overexpressing the BK_Ca α-subunit, H₂O₂ inhibits BK_Ca channel currents by oxidizing the cysteine residues on the α- subunit.^36–38 In the present study, H₂O₂ had no inhibitory effect on BK_Ca channel currents in inside-out patches. The reasons for these discrepancies remain unclear but could be related to variation of channels in native cells versus over-expression systems or the H₂O₂ concentrations (50 µmol/L used in the current study, and < 1 mmol/L H₂O₂ used in others).^36–38 In HCAs, the dilation induced by the highest concentration of H₂O₂ (300 µmol/L) was blocked by high K⁺ but not affected by the BK_Ca channel blocker iberiotoxin, indicating the possible involvement of other K⁺ channels. The identities of such K⁺ channels remain to be determined.

In patch-clamp studies, we observed that, in some cell-attached patches, BK_Ca channels exhibit smaller single-channel conductance (e.g., 100–200 pS) even with high-K⁺ bath solution to nullify effects of cell resting membrane potential. Our results are comparable to those obtained in smooth muscle cells from large human coronary arteries.²² The reasons for the reduced conductance are unclear. Because the K⁺ currents recorded in cell-attached patches are sensitive to the specific BK_Ca blocker paxilline and the conductance of these currents increases to approximately 240–250 pS once the patch is detached from the cell to form an inside-out patch, we suggest that the properties of the currents studied are most consistent with those of BK_Ca channels.

Study limitations

Exogenous H₂O₂ has been widely used to study the role of H₂O₂ in cell signaling.² In the present study, EC₅₀ (the concentration of drug that produces a 50% maximal response) of exogenous H₂O₂-induced dilation in HCAs is approximately 3×10⁻⁵ mol/L, with a minimum concentration required to induce a catalase-sensitive vasodilation of approximately 10⁻⁵ mol/L. These concentrations, similar to or lower than those used in other studies,^{7–11,26–32} are higher than those reported for endogenously generated H₂O₂ (<10⁻⁶ mol/L) by physiological stimuli such as shear stress⁴ and A23187.³⁹ This could be explained by the fact that the intracellular H₂O₂ may only reach 1–15% of the exogenously applied concentration,² probably due to endogenous anti-oxidant systems that protect cells from ROS. It is also possible that H₂O₂ is generated locally at higher concentrations than those measured, before it diffuses to the active site.

Using immunoblotting and immunocytochemistry, we found that H₂O₂ induced PKG-Iα dimerization and translocation to the plasma membrane in coronary SMCs. The dimerization results in the activation of PKG-Iα associated with a decrease in the enzyme’s K_m value.¹⁴ It is presumed, though not directly tested, that H₂O₂-induced dimerization of PKG-Iα contributes to the opening of BK_Ca channels and smooth muscle dilation in HCAs. Because there are no specific pharmacological agents available to inhibit the dimerization of PKG-Iα by H₂O₂, testing this hypothesis would require genetic manipulation of cysteine residues of PKG-Iα,¹⁴ an approach not feasible for isolated human blood vessels. Similarly, we did not directly assess PKG-Iα-mediated protein phosphorylation of BK_Ca channels, the result of which could provide further support for the activation of BK_Ca by dimerized PKG-Iα. Since specific antibodies for PKG-targeted residue in BK_Ca (serine 1072) are not available, the results obtained with a general anti-phosphoserine antibody may be difficult to interpret because other protein kinases (such as PKC) also phosphorylate serine residues of the BK_Ca channel.

Clinical implications

Flow-induced dilation of coronary arterioles from patients with CAD is mediated by a unique mechanism requiring the release of H₂O₂ from endothelial cells and subsequent smooth muscle hyperpolarization.^4–6 In the absence of CAD or its risk factors, other traditional mediators (i.e., NO and PGI₂) may play a more prominent role in dilation of coronary arterioles.⁶ In the present study, we provide evidence that the dimerization of PKG-Iα and subsequent activation of BK_Ca channels are two key intracellular signaling events responsible for H₂O₂-induced smooth muscle hyperpolarization and relaxation, thus revealing a fundamental signaling mechanism responsible for the role of H₂O₂ in regulation of coronary vascular tone and blood flow. With near maximal cardiac extraction of oxygen at rest, it is essential that myocardial perfusion is closely coupled to increases in myocyte metabolism.²³ By serving as a potent vasodilator in the coronary microcirculation, H₂O₂, derived from the blood vessel itself as well as from beating myocytes, may be an important endogenous regulator of myocardial perfusion under normal conditions and in disease states.²³

Novelty and Significance.

What Is Known?

• Hydrogen peroxide (H₂O₂) is a relatively stable reactive oxygen species (ROS) that serves as a signaling molecule in diverse physiological and pathophysiological responses.

• H₂O₂ is a key transferable endothelium-derived mediator responsible for flow-induced dilation and to a lesser extent receptor agonist-induced dilation in the human coronary microcirculation, particularly in patients with or at risk for coronary artery disease.

• H₂O₂ dilates blood vessels through the activation of smooth muscle K⁺ channels, including the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel.

What New Information Does This Article Contribute?

• Exogenous H₂O₂ opens arterial smooth muscle BK_Ca channels resulting in dilation of human coronary arterioles.

• The mechanism by which H₂O₂ opens BK_Ca channels involves dimerization and translocation of protein kinase G (PKG)-Iα.

• A similar signaling pathway, involving BK_Ca activation via PKG-Iα, mediates flow-induced and H₂O₂-dependent dilation of human coronary arterioles.

Accumulating evidence suggests that H₂O₂ serves as an important signaling molecule in the vascular system, although it is traditionally viewed as a harmful by-product of cell metabolism. Endothelial H₂O₂, is the key diffusible factor mediating flow-induced (and to a lesser extent bradykinin-induced) dilation in the human coronary microcirculation. However, the precise mechanisms by which H₂O₂ relaxes coronary arterioles remain unclear. In this study, we found that exogenous H₂O₂ dilates human coronary arterioles by opening smooth muscle BK_Ca channels through dimerization and translocation of PKG-Iα. Flow or shear stress, which releases endothelium-derived H₂O₂, dilates coronary arterioles by activating a signaling pathway similar to that induced by exogenous H₂O₂. Our findings provide further support for the proposed role of H₂O₂ as a diffusible endothelium-derived hyperpolarizing factor in both animals and humans. and have implications for antioxidant treatment strategies in patients with coronary disease.

Non-standard abbreviations and acronyms

cAMP

adenosine 3',5'-cyclic monophosphate

3-AT

3-amino-1,2,4-triazole

cGMP

guanosine 3’,5’-cyclic monophosphate

BK_Ca channel

large conductance Ca²⁺-activated K⁺ channel

CAD

coronary artery disease

EC

endothelial cell

EDHF

endothelium-derived hyperpolarizing factor

FMD

flow-mediated dilation

HCA

human coronary arteriole

HCASMC

human coronary artery smooth muscle cell

H₂O₂

hydrogen peroxide

IbTX

iberiotoxin

K_ATP channel

ATP-sensitive K⁺ channel

K_Ca channel

Ca²⁺-activated K⁺ channel

Kv channel

voltage-gated K⁺ channel

L-NAME

N^G-nitro-L-arginine methyl ester

NPo

open state probability

ODQ

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one

PKA

protein kinase A or cAMP-dependent protein kinase

PKC

protein kinase C

PKG

protein kinase G or cGMP-dependent protein kinase

PSS

physiological salt solution

ROS

reactive oxygen species

sGC

soluble guanylate cyclase

SMC

smooth muscle cell